Gel electrophoresis 08

Gel ElectrophoresisGel Electrophoresis

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DefinitionDefinition

Gel electrophoresis is a technique used to separate DNA bands based on size

Quick QuizQuick Quiz

Gel electrophoresis separates molecular products based on:

A. ShapeB. SizeC. ChargeD. Evolutionary similarity

ExamplesExamples

Gel Electrophoresis is used to visualize…

Genomic DNARNAPCR productsPlasmidsRestriction enzyme

digest productshttp://www.steve.gb.com/images/science/agarose.jpg

Non-exampleNon-example

Gel Electrophoresis is not used to…

Separate DNA from the cell or from tissues

AgaroseAgarose

Neutral polysaccharide

Harvested from Rhodophyceae algal cell wallshttp://www.steve.gb.com/images/

AgaroseAgarose Alternating sugar units

form linear chains Cross-linking chains via

hydrogen bonds forms porous matrix

Heat breaks matrix apart

Cooling forms a matrix with an “average” pore size

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When traveling through an agarose gel, larger molecular products will migrate _________ smaller molecular products.

A. Faster thanB. Slower thanC. At the same rate

Why Run a Gel?Why Run a Gel?

Molecular weight markers are DNA fragments of known size

Comparison of sample bands to markers allows… Visible confirmation of

desired product

Quantification of sample DNA

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http://www.sumanasinc.com/webcontent/anisamples/majorsbiology/gelelectrophoresis.html

http://learn.genetics.utah.edu/units/biotech/gel

How Gel Electrophoresis WorksHow Gel Electrophoresis Works












As DNA has a net _______ charge, it migrates towards the gel box’s _________.

A. Positive; cathodeB. Positive; anodeC. Negative; cathodeD. Negative; anode

Visualizing DNAVisualizing DNA

Ethidium Bromide binds to DNA

Fluorescence under UV light makes bands visible

http://www.lanl.gov/news/albums/bioscience/AnthraxUVplate.sized.jpg


The size of the molecular products is determined by:

A. The intensity of the bandB. The percentage of agarose gelC. Comparison with a molecular weight “ladder”D. Being familiar with your gel and “eyeballing” it

Gel Electrophoresis Gel Electrophoresis

Prepare agarose gelMelt, cool and add Ethidium Bromide. Mix thoroughly.

Add running buffer, load samples and marker

Run gel at constant voltage until band separation occurs

Pour into casting tray with comb and allow to solidify

View DNA on UV light box and document results


Buffer is used instead of water when making and running gels because:

A. Buffer enhances the transmission of electric currents in water

B. Buffer is more homogeneous than waterC. Buffer neutralizes the charge on molecular products

prior to electrophoresisD. Buffers can be customized based upon the

electrophoresis protocol

Troubleshooting GuideTroubleshooting Guide

DNA Science: A First Course in Recombinant DNA Technology, by David A. Micklos and Greg A. Freyer

ENDEND

ResourcesResources

Gel Electrophoresis Handout

DNA Science: A First Course in Recombinant DNA Technology, by David A. Micklos and Greg A. Freyer





California State Chemistry Standards California State Chemistry Standards

Grades 9-12

2. Properties of matter result from the ability of atoms to form bonds…

6. Solutions are homogenous mixtures of two or more substances

10b. Students know the bonding characteristics of carbon…

California State Biology StandardsCalifornia State Biology Standards

Grades 9-12

5d. Students know the general structure and function of DNA

California State California State Investigation and ExperimentationInvestigation and Experimentation

StandardsStandards

Grade 7

a. Select and use appropriate tools and technology to perform tests, collect data, analyze relationships, and display data

Grades 9-12

c. Identify possible reasons for inconsistent results, such as sources of error or uncontrolled conditions

d. Formulate explanations by using logic and evidence

k. Recognize the cumulative nature of scientific evidence

l. Analyze situations and solve problems that require combining and applying concepts from more than one area of science

National StandardsNational Standards

Grades 6-12

Content Standard A: Science as Inquiry

Content Standard C: Life Science

Content Standard E: Science and Technology